Plant materials and growth conditions
C. intermedia seeds were collected from Helin County, Hohhot, Inner Mongolia, China. No specific field permissions were required to collect the plant samples and seeds. The full, intact and healthy seeds were selected and routinely grown in a 1:3 mixture of peat soil and vermiculite in a greenhouse (22 °C, 16 h light/8 h dark). For transient expression assays, fully expended leaves of three-week-old seedlings were chosen for injection (Additional file 1: Figure S1a).
Agrobacterium strains and plasmids
The Agrobacterium tumefaciens strains, GV3101, EHA105, EHA101, LBA4404 and AGL1 were purchased from Shanghai Weidi Biotechnology Co, Ltd. The detailed information of all the five Agrobacterium strains were listed in Table 1. The pCAMBIA1305.2 binary vector (kept by the Key Laboratory of Plant Stress Physiology and Molecular Biology, Inner Mongolia Agricultural University) which contains the CaMV35S promoter, GUSPlus reporter gene, kanamycin resistance gene for bacteria selection and hygromycin resistance gene for plant selection, was transformed into five Agrobacterial strains by electroporation, respectively. The expression of both GUSPlus reporter gene and kanamycin resistance gene were driven by CaMV35S promoter and the GUSPlus reported gene expressed only in plant cells but not in bacteria cells.
Agrobacterium cell culture and preparation of infiltration suspension
A single colony of A. tumefaciens strains GV3101, EHA105, EHA101, LBA4404 and AGL1 harboring pCAMBIA1305.2 binary vector was inoculated in 4 mL Luria Broth (LB) liquid medium (50 μg/mL kanamycin and 25 μg/mL gentamicin for GV3101, 50 μg/mL kanamycin and 20 μg /mL rifampicin for EHA105, EHA101, LBA4404 and AGL1), and was incubated overnight at 28 °C at 200 rpm. On the next day, an aliquot of 1.5 mL Agrobacterial cells were transferred to 25 mL fresh LB liquid medium (1:25 ratio, v/v) supplemented with kanamycin (50 μg /mL) and gentamicin (25 μg /mL) for GV3101, or kanamycin (50 μg /mL) and rifampicin (20 μg /mL) for other trains. All the strains were allowed to grow until the cell density reached an OD600 of 1.3. The cells were centrifuged at 5000 ~ 6000 rpm for 10 min at 4 °C and the supernatant was discard. The cells were resuspended with the infiltration medium (1/2MS medium (PH5.8), 10 mmol/L MES, 10 mmol/L MgCl2, 100 μmol /L acetosyringone) to OD600 = 0.7~0.8. To optimize the infiltration condition, several surfactants such as Silwet L-77, Tween-20 and Triton X-100 were added to the infiltration medium, and various concentrations with 0.001% (v/v), 0.005% (v/v) or 0.01% (v/v) of the surfactant were optimized based on the optimal surfactant. Prior to infiltration, the Agrobacterial cells suspensions were incubated at room temperature under darkness for 3~4 h since the Vir gene in Agrobacterial cells need to be fully induced by acetosyringone, and during this time, the Agrobacterial cells suspensions should be mixed upside down every half hour since the Agrobacterial cells could be uniformly suspended in the 1/2 MS medium.
Infiltration of the C. intermedia leaves
The 3-week-old seedlings was chosen to infiltrate (We chose 3-week-old plants because the leaves of C. intermedia were relatively young and fully expanded after growing for about three weeks, which are pretty beneficial for transient expression in C. intermedia.). The Agrobacterial cells suspensions was pressure-infiltrated to the abaxial surface of C. intermedia leaves using a 1 mL disposable needleless syringe (Additional file 1: Figure S1b). Infiltrating leaves in sections until the whole area appears translucent, and leaves were saturated with Agrobacterial cells suspensions should be separated and should not touch each other (Additional file 1: Figure S1c). Immediately after infiltration, the plant was covered with dark plastic film for 24 h to maintain a high relative humidity (> 80%), and was kept in dark condition for another 2 days, then the plants were grown in the normal condition (22 °C, 16 h light/8 h dark). Last but not the least, it is necessary to spray ddH2O to the plants in the growth chamber, by doing this, the relative humidity of plants surrounding was high and the efficiency of transient expression was improved . The untransformed C. intermedia seedlings were used as the negative control.
β-Glucuronidase (GUS) histochemical staining
The C. intermedia leaves were collected in 2~11 days after infiltration and 10~12 leaves from each experimental condition were taken each day. Histochemical GUS staining was carried out as described previously . The infiltrated leaves were submerged in the GUS staining solution (50 mM NaH2PO4-Na2HPO4, pH 7.3, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6, 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 0.1% (v/v) Triton X-100) at 37 °C for 6~16 h. Then the stained leaves were rinsed with 95% ethanol (at least 3 times, each time 15 min) in a high temperature (above 90 °C) to remove chlorophylls from plant tissues until the leaves were totally cleared and photographed.
RNA extraction and real time RT-PCR assay
For the total RNA extraction, the C. intermedia leaves injected with A. tumefaciens transformed with pCanG-HA empty vector or CiDREB1C (GenBank: MG748598) were harvested at various time points, including 0, 2, 3, 4, 5, 6, 7, 8, 9, 12, 15, 18, 21, 24 and 27 days after infiltration. Total RNA was isolated from each sample according to the manufactures’ instruction (Invitrogen) of TRIzol reagent. About 2 μg of total RNA was reverse-transcribed into cDNA with oligo (dT)18 primers according to TransScript gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China, Cat# AT311). For real-time PCR analysis, the cDNA was diluted 16-fold with sterile DEPC water and 5 μL was added into a 20 μL PCR reaction. The real-time PCR was performed using SYBR Green I Master (Roche) on a LightCycler 480 system (Roche, Basel, Switzerland), which the following cycling parameters: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 15 s. A melting curve was generated for each sample at the end of running to access the purity of amplified products. CiEF1α (GenBank: KC679842) gene was used as internal references to normalize the samples. The expression level was calculated from the cycle threshold based on the 2-ΔΔCT and 2-ΔCT methods, with three technical replicates were performed at each experiment and at least two independent repetitions of the biological experiments were performed. The primers used in this study were listed in Additional file 5: Table S1.
Stress resistance detection of transiently transformed in C. intermedia
Drought tolerance was detected mainly according to Wan et al. , and with slightly modification. Plants were grown in soil for about 20 days with sufficient watering. Then, the plant leaves were infiltrated with A. tumefaciens containing pCanG-HA empty vector or CiDREB1C gene. After infiltrated for 2 days, the plants were subjected to drought treatment by halting irrigation, and the plants infiltrated with pCanG-HA empty vector were used as control. Every pot was placed under the same conditions and the position of each pot was changed randomly every day. When the seedlings showed obvious wilting, yellowing and lethal effects of dehydration, the watering was restored. The survival rate of each line was calculated and the total chlorophyll content was measured after 8 days of re-watering. In the above experiment, there was four plants in each pot and at least four pots for both control and transient expression CiDREB1C seedlings. The photographs were taken immediately for at 17 days after drought treatments and at 4 days or 8 days after re-watering. The experiments were carried out with three biological replicates.
For salt stress, we mainly referred to Han et al.  and Wan et al. , and with minor modifications. Briefly, after infiltrated for 3 days, the plants were irrigated with 250 mmol/L NaCl solution and those infiltrated with pCanG-HA empty vector were taken as control. We poured about 2~3 L NaCl solution into the big tray containing pots with seedlings, which allowed each pot to take up enough NaCl solution from bottom to top naturally, and then poured out the excess NaCl solution after 24 h soaking. The NaCl solution was watered every 4~5 days until the seedlings showed apparent phenotype, the survival rate of each line was calculated, and the total chlorophyll content and seedlings growth rate were measured. The seedlings growth rate is the ratio of the height of seedlings growth during treatment to the total height of seedlings, which could reflect growth status and growth rate of seedlings under different stresses. The photographs were taken immediately at 11 and 21 days respectively after NaCl treatments. The experiments were carried out with three biological replicates.
For ABA treatment, the plants were sprayed with ABA aqueous solution (100 μmol/L, with 0.01% Silwet L-77) and the plants infiltrated with pCanG-HA empty vector were taken as control. We spray ABA solution on the leaves of each plant until the surface of leaves were all covered with ABA solution. The ABA aqueous solution was sprayed 4~5 times a day until the seedlings leaves showed obvious detachment and wilting phenotype, the survival rate and leaf drop rates of each line was calculated, and the total chlorophyll content was measured. The photographs were taken immediately at 4 or 10 days after ABA treatments. The experiments were also carried out with three biological replicates.